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Resistance to angiotensin II (Ang II)-induced hypertension in T-cell deficient male mice with a targeted mutation in the recombination activating gene-1 (Rag1) on the C57BL/6J background (B6.Rag1−/−-M), which was reported by five independent laboratories including ours prior to 2015, has been lost. In mice purchased from Jackson Laboratory in 2015 and 2016, the time course and magnitude increase in mean arterial pressure (MAP) induced by two weeks of Ang II infusion at 490 ng/kg/min was identical between B6.Rag1−/−-M and wildtype littermates (B6.WT-M). Moreover, there were no differences in the time course or magnitude increase in MAP at the lowest dose of Ang II (200 ng/kg/min) that increased MAP. This loss in Ang II resistance is independent of T-cells. Angiotensin-type1-receptor (AT1R) binding was 1.4-fold higher in glomeruli isolated from recently purchased B6.Rag1−/−-M suggesting an increase in renal AT1R activity masks the blood pressure protection afforded by the lack of T-cells. The phenotypic change in B6.Rag1−/−-M has implications for investigators using this strain to study mechanisms of T-cell modulation of Ang II-dependent blood pressure control. These findings also serve as a reminder that the universal drive for genetic variation occurs in all animals including inbred mouse strains and that spontaneous mutations leading to phenotypic change can compromise experimental reproducibility over time and place. Lastly, these observations illustrate the importance of including experimental details regarding the location and time period over which animals are bred in publications involving animal studies to promote rigor and reproducibility in the scientific literature.

Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5= untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. Ezrin is a prototype member of the ERM (ezrin-radixin-moesin) family of proteins that functions as a scaffold between the plasma membrane and the underlying cortical actin cytoskeleton (1, 2). Ezrin regulates cytoskeletal dynamics in response to both internal and external stimuli through its intracellular localization and protein binding activities; thus, it plays an important role in the maintenance of cell shape, cell polarity, adhesion, and movement (3). All members of the ERM family are characterized by the presence of a shared FERM domain at the amino terminus, which can bind to transmembrane proteins, including CD43, CD44, CD95, ICAMs, syndecan 2, EBP50/NHERF1, and E3KARP/NHERF2. The carboxy termini of ERM proteins contain an F-actin binding domain, which both regulates intramolecular interactions with amino-terminal FERM domains and promotes F-actin organization. The pleiotropic functions of ezrin in a wide range of cellular processes can be explained through its association with numerous proteins with diverse functions (4). Several lines of evidence have indicated that ezrin can oscillate between various "open/active" and "closed/dormant" states, which are regulated by self-association of N-terminal and Cterminal regions. Head-to-tail folding of the molecule likely masks the respective protein binding sites and leads to the localization of ezrin in the cytoplasm in its monomeric form. The conformational switch to an open state requires direct interaction with the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and phosphorylation of a conserved threonine (T567) located in the ...